Type 2 adenovirus DNA can be cleaved with a combination of site- specific restriction endonucleases into 25 or more ordered fragments ranging from 4200 to 170 base pairs in length. The strands of many fragments can also be separated by gel electrophoresis after alkaline denaturation. The fragments and the separated strands can be used to construct a detailed replication map. In addition to merely locating origins and termini for replication, the map can indicate whether or not synthesis occurs by a strand displacement mechanism and if replication proceeds through the ends of the chromosome. In fact, adenovirus replication may be quite complicated; a preliminary map reveals several origins at internal positions as well as at both ends of the molecule. The role and control ofthese initiation sites are not yet known. The mechanism ofchain elongation will be studied using ethidium bromide and hydroxyurea, specific inhibitors of adenovirus replication. In particular, small, transient chains can be identified during inhibition. The strand-specificity, chromosomal location, and fate of the chains will be determined by hybridization. A model for the structure of the inverted terminal repetition has been developed. The model allows the 3' and 5' ends of each strand to come close together in a limited duplex region. Ligase or a combination of DNA polymerase and ligase can then join the ends to form a covalently $ closed, circular template for replication. In vitro circle formation will be monitored by biophysical methods as well as electron microscopy.